The methyltransferase N6AMT1 participates in the cell cycle by regulating cyclin E levels.

The methyltransferase N6AMT1 has been associated with the progression of different pathological conditions, such as tumours and neurological malfunctions, but the underlying mechanism is not fully understood. Analysis of N6AMT1-depleted cells revealed that N6AMT1 is involved in the cell cycle and cell proliferation. In N6AMT1-depleted cells, the cell doubling time was increased, and cell progression out of mitosis and the G0/G1 and S phases was disrupted. It was discovered that in N6AMT1-depleted cells, the transcription of cyclin E was downregulated, which indicates that N6AMT1 is involved in the regulation of cyclin E transcription. Understanding the functions and importance of N6AMT1 in cell proliferation and cell cycle regulation is essential for developing treatments and strategies to control diseases that are associated with N6AMT1.

A sensing strategy combining T7 promoter-contained DNA probe with CRISPR/Cas13a for detection of bacteria and human methyltransferase.

Abnormal DNA methylation is closely related to the occurrence and development of many diseases. The determination of human DNA methyltransferase activity and the screening of its inhibitors are extreme important for the diagnosis and the treatment of methylation-related diseases in clinic. Most of the current detection methods have the disadvantages of sophisticated design, high cost and low detection limit. By combining T7 promoter-contained DNA probe as the substrate for methyltransferase with CRISPR/Cas13a sensing strategy, a novel fluorescent sensing platform is designed to achieve simple, specific, sensitive detection of bacteria DNA methyltransferase (DNA-(N-6-adenine)-methyltransferase, Dam MTase) and also human methyltransferase (DNA (cytosine-5)-methyltransferase 1, Dnmt1). A hairpin DNA probe designed for Dam MTase and a double strand DNA probe for Dnmt1 are both methylated followed by the methylation-dependent site-specific cleavage, which result a T7 promoter-contained product and a T7 promoter-free one to respectively open and close the transcription and subsequent CRISPR/Cas13a target-initiated cleavage of fluorescence-labeled reporter RNA. In virtue of the specificity of methylation-dependent cleavage of probe, the efficient transcription amplification and CRISPR/Cas13a sequence-specific sensing, this strategy exhibited remarkable specificity and sensitivity, with the limit of detection of 3.10 × 10-5 U/mL for Dam MTase. Moreover, Dnmt1 activity in MCF-7 cells was detected and the inhibition of Apt. #9 was evaluated. This strategy for methyltransferase detection is convenient and efficient for inhibitor discovery and early cancer diagnosis.

Reducing N6AMT1-mediated 6mA DNA modification promotes breast tumor progression via transcriptional repressing cell cycle inhibitors.

DNA N6-methyladenosine (6mA) is a novel epigenetic signaling modification in humans and has been implicated in the progression and tumorigenesis of several cancers. However, the function and mechanism of 6mA in breast cancer (BC), the most common cancer among women, are unclear. Here, we found that decreases in N6AMT1 correlated with the extent of 6mA in clinical BC tissues and predicted a worse survival of BC patients. Functionally, knockdown of N6AMT1 markedly reduced 6mA in DNA and promoted colony formation and migration of BC cells, whereas overexpression of N6AMT1 had the opposite effect. Moreover, silencing of N6AMT1 reduced 6mA modification and enhanced the growth of BC cells in vitro and tumors in vivo. 6mA immunoprecipitation sequencing (6mA-IP-seq), RNA-seq, 6mA-IP-PCR, and bioinformatics analysis indicated that N6AMT1 was a functional methyltransferase for genomic 6mA DNA modifications and related to gene transcriptional activity. Critical negative regulators of the cell cycle, such as RB1, P21, REST, and TP53 were identified as targets of N6AMT1 in BC. These results suggest N6AMT1 enhances DNA 6mA levels to repress tumor progression via transcriptional regulation of cell cycle inhibitors.

The enhanced genomic 6 mA metabolism contributes to the proliferation and migration of TSCC cells.

In contrast to the well-established genomic 5-methylcytosine (5mC), the existence of N6-methyladenine (6 mA) in eukaryotic genomes was discovered only recently. Initial studies found that it was actively regulated in cancer cells, suggesting its involvement in the process of carcinogenesis. However, the contribution of 6 mA in tongue squamous cell carcinoma (TSCC) still remains uncharacterized. In this study, a pan-cancer type analysis was first performed, which revealed enhanced 6 mA metabolism in diverse cancer types. The study was then focused on the regulation of 6 mA metabolism, as well as its effects on TSCC cells. To these aspects, genome 6 mA level was found greatly increased in TSCC tissues and cultured cells. By knocking down 6 mA methylases N6AMT1 and METTL4, the level of genomic 6 mA was decreased in TSCC cells. This led to suppressed colony formation and cell migration. By contrast, knockdown of 6 mA demethylase ALKBH1 resulted in an increased 6 mA level, enhanced colony formation, and cell migration. Further study suggested that regulation of the NF-κB pathway might contribute to the enhanced migration of TSCC cells. Therefore, in the case of TSCC, we have shown that genomic 6 mA modification is involved in the proliferation and migration of cancer cells.

DNA N6-methyladenine involvement and regulation of hepatocellular carcinoma development.

DNA N6-methyladenine (6 mA) is a new type of DNA methylation identified in various eukaryotic cells. However, its alteration and genomic distribution features in hepatocellular carcinoma (HCC) remain elusive. In this study, we found that N6AMT1 overexpression increased HCC cell viability, suppressed apoptosis, and enhanced migration and invasion, whereas ALKBH1 overexpression induced the opposite effects. Further, 23,779 gain-of-6 mA regions and 11,240 loss-of-6 mA regions were differentially identified in HCC tissues. The differential gain and loss of 6 mA regions were considerably enriched in intergenic regions. Moreover, 7% of the differential 6 mA modifications were associated with tumors, with 60 associated with oncogenes and 57 with tumor suppressor genes (TSGs), and 17 were common to oncogenes and TSGs. The candidate genes affected by 6 mA were filtered by gene ontology (GO) and RNA-seq. Using quantitative polymerase chain reaction (qPCR), BCL2 and PARTICL were found to be correlated with DNA 6 mA in certain HCC processes.

KMT9 Controls Stemness and Growth of Colorectal Cancer.

Colorectal cancer is among the leading causes of cancer-associated deaths worldwide. Treatment failure and tumor recurrence due to survival of therapy-resistant cancer stem/initiating cells represent major clinical issues to overcome. In this study, we identified lysine methyltransferase 9 (KMT9), an obligate heterodimer composed of KMT9α and KMT9ß that monomethylates histone H4 at lysine 12 (H4K12me1), as an important regulator in colorectal tumorigenesis. KMT9α and KMT9ß were overexpressed in colorectal cancer and colocalized with H4K12me1 at promoters of target genes involved in the regulation of proliferation. Ablation of KMT9α drastically reduced colorectal tumorigenesis in mice and prevented the growth of murine as well as human patient-derived tumor organoids. Moreover, loss of KMT9α impaired the maintenance and function of colorectal cancer stem/initiating cells and induced apoptosis specifically in this cellular compartment. Together, these data suggest that KMT9 is an important regulator of colorectal carcinogenesis, identifying KMT9 as a promising therapeutic target for the treatment of colorectal cancer. SIGNIFICANCE: The H4K12 methyltransferase KMT9 regulates tumor cell proliferation and stemness in colorectal cancer, indicating that targeting KMT9 could be a useful approach for preventing and treating this disease.

Ultra-sensitive detection of DNA N6-adenine methyltransferase based on a 3D tetrahedral fluorescence scaffold assisted by symmetrical double-ring dumbbells.

DNA methylation is an epigenetic modification that plays a vital role in X chromosome inactivation, genome imprinting, and gene expression. DNA methyltransferase establishes and maintains a stable methylation state in genomic DNA. Efficient and specific DNA methyltransferase testing is essential for the early diagnosis and treatment of cancer. In this study, we designed an ultra-sensitive fluorescent biosensor, based on a 3D tetrahedral fluorescent scaffold assisted by symmetrical double-ring dumbbells, for the detection of DNA-[N 6-adenine]-methyltransferase (Dam MTase). Double-stranded DNA was methylated by Dam MTase and then digested by DpnI to form two identical dumbbell rings. The 3D tetrahedral fluorescent scaffold was synthesized from four oligonucleotide chains containing hairpins. When the sheared dumbbells reacted with the 3D tetrahedral fluorescent scaffold, the hairpins opened and a fluorescence signal could be detected. The strategy was successful over a wide detection range, from 0.002 to 100 U mL-1 Dam MTase, and the lowest detection limit was 0.00036 U mL-1. Control experiments with M.SssI methyltransferase and HpaII methylation restriction endonuclease confirmed the specificity of the method. Experiments with spiked human serum and the 5-fluorouracil inhibitor proved the suitability of the method for early cancer diagnosis.

Clostridioides difficile specific DNA adenine methyltransferase CamA squeezes and flips adenine out of DNA helix.

Clostridioides difficile infections are an urgent medical problem. The newly discovered C. difficile adenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-L-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.

Highly sensitive detection of DNA methyltransferase activity and its inhibitor screening by coupling fluorescence correlation spectroscopy with polystyrene polymer dots.

DNA methylation is a critical part of epigenetics and plays a vital role in maintaining normal cell function, genetic imprinting, and human tumorigenesis. Thus, it is important to develop a sensitive method for the determination of DNA methyltransferase (MTase) activity. Here, we present a simple and sensitive method based on single molecule fluorescence correlation spectroscopy (FCS) and polystyrene polymer dots (PS Pdots) for the quantitative detection of DNA adenine methylation (Dam) MTase activity and its inhibitor screening in homogeneous solution without separation. Its principle is based on the measurement of the characteristic diffusion time (τD) of unmethylated and methylated DNA-fluorescent probes by FCS. A hairpin DNA probe including the 5'-GATC-3' sequence is used by doubly labelling fluorophore Alexa Fluor 488 (Alexa 488) and biotin at the 5'- and 3'-terminus, respectively. Dam MTase catalyzed the methylation of the sequence of 5'-GATC-3', and DpnI cleaved the sequence of 5'-G-Am-TC-3'. Streptavidin conjugated PS Pdots were used to react with DNA probes without methylation to further increase the difference in τD values between methylated and unmethylated DNA-Alexa 488 probes. We used the FCS method to measure the τD values of DNA-Alexa 488 probes and further obtained the activity of Dam MTase. It is found that the τD value of the methylated DNA probe is negatively correlated with the logarithm of Dam MTase concentration in the range from 0.025 U mL-1 to 3 U mL-1. The detection limit is as low as 0.025 U mL-1. Furthermore, we evaluated the inhibition effect of drug-related DNA methylation and the half-maximal inhibitory concentration (IC50) value is consistent with a previous study. The results demonstrated that our proposed method will become a promising platform for the determination of Dam MTase activity and inhibitor screening.

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation.

BACKGROUND: Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear. RESULTS: A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inflammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inflammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1, secretion of IL-1ß and phosphorylation of c-Jun N-terminal kinase (Jnk). Transcriptome sequencing (RNA-seq) results showed that most of the differentially expressed genes and enriched KEGG pathways between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups overlapped, which includes multiple signaling pathways related to the inflammasome. C50336Δdam and C50336::dam were both found to be defective in suppressing the expression of several anti-inflammasome factors. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically enhance the activation of NLRP3 inflammasome independently via promoting the Jnk pathway. CONCLUSIONS: These data indicated that Dam was essential for modulating inflammasome activation during SE infection, there were complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

Methyl Adenine Identification (MadID): High-Resolution Detection of Protein-DNA Interactions.

Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understand many essential biological processes. Methyl Adenine Identification (MadID) is a proximity methylation-based assay that allows the visualization, quantification, and identification of binding sites from DNA-interacting proteins in eukaryotic cells. Chromatin-binding proteins of interest are fused to the newly described bacterial methyltransferase M.EcoGII. This enzyme catalyzes the methylation of adenine residues with no sequence specificity. Consequently, adenines within and in the vicinity of the protein binding sites will be decorated with a methyl group (m6A), a modification that can be further detected using different methods. M.EcoGII-dependent DNA methylation can be monitored in situ using immunostaining, at the genome-wide level using a combination of m6A-specific immunoprecipitation and whole-genome sequencing, or locally at DNA regions of interest purified by chromatin immunoprecipitation or probe-based capture techniques. MadID is conceptually similar to DNA adenine methyltransferase identification (DamID) that relies on the methylation of GATC motifs. However, MadID provides a higher resolution, deeper coverage, and opens ways for identification of binding sites in genomic regions that were largely inaccessible such as telomeres, centromeres, and repeated elements.

Terminal deoxynucleotidyl transferase induced activators to unlock the trans-cleavage of CRISPR/Cpf 1 (TdT-IU- CRISPR/Cpf 1): An ultrasensitive biosensor for Dam MTase activity detection.

DNA adenine methylation methyltransferase (Dam MTase) plays an important role in gene expression and cell development, and involves in some development of tumors. There are many methods to detect Dam MTase activity, yet they have some shortcomings such as high cost, limited detection limit and complicated operation. Here, a new fast and simple multiple amplification strategy based on terminal deoxynucleotidyl transferase induced activators to unlock the collateral cleavage activities (trans-cleavage) of CRISPR/Cpf 1 (TdT-IU-CRISPR/Cpf 1) was firstly established. Based on this, a new fluorescent biosensor for Dam MTase activity detection was proposed. Importantly, the proposed biosensor does not require strict control over the temperature changes, and has ultrasensitive detection with limit of detection as low as 1.26 × 10-3 U/mL. Moreover, the novel biosensor can not only be applied for screening the inhibitors for Dam MTase activity, but also can detect its activity in complex biological samples. The newly established multiple amplification strategy TdT-IU-CRISPR/Cpf 1 and the proposed biosensor for Dam MTase activity will be of great significance in biomedical testing and clinical diagnostics.

Simultaneous quantification of protein-DNA interactions and transcriptomes in single cells with scDam&T-seq.

Protein-DNA interactions are essential for establishing cell type-specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a multi-omics method that can simultaneously quantify protein-DNA interactions and the transcriptome in single cells. The method effectively combines two existing methods: DNA adenine methyltransferase identification (DamID) and CEL-Seq2. DamID works through the tethering of a protein of interest (POI) to the Escherichia coli DNA adenine methyltransferase (Dam). Upon expression of this fusion protein, DNA in proximity to the POI is methylated by Dam and can be selectively digested and amplified. CEL-Seq2, in contrast, makes use of poly-dT primers to reverse transcribe mRNA, followed by linear amplification through in vitro transcription. scDam&T-seq is the first technique capable of providing a combined readout of protein-DNA contact and transcription from single-cell samples. Once suitable cell lines have been established, the protocol can be completed in 5 d, with a throughput of hundreds to thousands of cells. The processing of raw sequencing data takes an additional 1-2 d. Our method can be used to understand the transcriptional changes a cell undergoes upon the DNA binding of a POI. It can be performed in any laboratory with access to FACS, robotic and high-throughput-sequencing facilities.

Methyltransferase DnmA is responsible for genome-wide N6-methyladenosine modifications at non-palindromic recognition sites in Bacillus subtilis.

The genomes of organisms from all three domains of life harbor endogenous base modifications in the form of DNA methylation. In bacterial genomes, methylation occurs on adenosine and cytidine residues to include N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C). Bacterial DNA methylation has been well characterized in the context of restriction-modification (RM) systems, where methylation regulates DNA incision by the cognate restriction endonuclease. Relative to RM systems less is known about how m6A contributes to the epigenetic regulation of cellular functions in Gram-positive bacteria. Here, we characterize site-specific m6A modifications in the non-palindromic sequence GACGmAG within the genomes of Bacillus subtilis strains. We demonstrate that the yeeA gene is a methyltransferase responsible for the presence of m6A modifications. We show that methylation from YeeA does not function to limit DNA uptake during natural transformation. Instead, we identify a subset of promoters that contain the methylation consensus sequence and show that loss of methylation within promoter regions causes a decrease in reporter expression. Further, we identify a transcriptional repressor that preferentially binds an unmethylated promoter used in the reporter assays. With these results we suggest that m6A modifications in B. subtilis function to promote gene expression.

The Common Partner of Several Methyltransferases TRMT112 Regulates the Expression of N6AMT1 Isoforms in Mammalian Cells.

Methylation is a widespread modification occurring in DNA, RNA and proteins. The N6AMT1 (HEMK2) protein has DNA N6-methyladenine as well as the protein glutamine and histone lysine methyltransferase activities. The human genome encodes two different isoforms of N6AMT1, the major isoform and the alternatively spliced isoform, where the substrate binding motif is missing. Several RNA methyltransferases involved in ribosome biogenesis, tRNA methylation and translation interact with the common partner, the TRMT112 protein. In this study, we show that TRMT112 regulates the expression of N6AMT1 isoforms in mammalian cells. Both isoforms are equally expressed on mRNA level, but only isoform 1 is detected on the protein level in human cells. We show that the alternatively spliced isoform is not able to interact with TRMT112 and when translated, is rapidly degraded from the cells. This suggests that TRMT112 is involved in cellular quality control ensuring that N6AMT1 isoform with missing substrate binding domain is eliminated from the cells. The down-regulation of TRMT112 does not affect the N6AMT1 protein levels in cells, suggesting that the two proteins of TRMT112 network, WBSCR22 and N6AMT1, are differently regulated by their common cofactor.

Structural insights into the critical DNA damage sensors DNA-PKcs, ATM and ATR.

ATM, ATR and DNA-PKCs are key effectors of DNA Damage response and have been extensively linked to tumourigenesis and survival of cancer cells after radio/chemotherapy. Despite numerous efforts, the structures of these proteins remained elusive until very recently. The resolution revolution in Cryo-EM allowed for molecular details of these proteins to be seen for the first time. Here we provide a comprehensive review of the structures of ATM, ATR and DNA-PKcs and their complexes and expand with observations springing from our own cryo-EM studies. These observations include a novel conformation of ATR and novel dimeric arrangements of DNA-PKcs.

KMT9 monomethylates histone H4 lysine 12 and controls proliferation of prostate cancer cells.

Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-ß-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.

A dual-response biosensor for electrochemical and glucometer detection of DNA methyltransferase activity based on functionalized metal-organic framework amplification.

DNA methylation is catalyzed by DNA methyltransferase (MTase) and concerned with many biological processes including pathogenesis of various human diseases. The monitoring of MTase activity is thus of great significance in disease diagnosis and drug screening. Herein, we developed a facile way to synthesize biocompatible invertase enzyme modified metal-organic framework (Invertase/MOF) materials, and explored its application in constructing a dual-response Dam MTase sensor for the first time. By using them as signal probes, in which high density of metal sites could be electrochemically detected and invertase could hydrolyze sucrose into glucose for generation of glucometer signal output, dual-response for accurate detection of Dam MTase was realized. In the presence of Dam MTase, the methylation of hairpin probe 1 (HP1) occurred and thus caused the cleavage of HP1 assisted by a restriction endonuclease (DpnI) to produce the binding sequences. The binding sequences then hybridized with the electrode-assembled HP2 to expose their sticky termini which sequentially hybridized with the Invertase/MOFs-tethered capture probes. Finally, the electrodes were incubated with a sucrose solution, followed by the separate electrochemical and glucometer detection. The present assay brought good performance which could detect Dam MTase activity as low as 0.001 U mL-1 with wide linear range and good selectivity against other cytosine MTase (M.SssI MTase). Moreover, it also owns ability to be potentially applied for the inhibitors screening by utilization of 5-fluorouracil as an inhibitor model. The results imply that our proposed method provides a convenient platform for early cancer diagnosis and therapeutic applications.

Multiple sealed primers-mediated rolling circle amplification strategy for sensitive and specific detection of DNA methyltransferase activity.

DNA methyltransferase (MTase) aberrant expression has a close relationship to tumorigenesis. DNA MTase activity detection is of great importance to its biomedical research and theranostics study. Here, multiple sealed primers-mediated rolling circle amplification (RCA) strategy is developed for sensitively and specifically detecting DNA MTase activity. The DNA probe has a folded, double-loop structure that seals multiple primers. First, in the presence of DNA MTase, the DNA probe is methylated, which then gets cleaved by the restriction endonuclease and breaks into multiple DNA oligonucleotide fragments. Second, each DNA oligonucleotide fragment acts as an independent primer for triggering RCA reaction respectively, producing long DNA strands that contain several interval G-quadruplexes. Finally, copious of G-quadruplexes are obtained, which bind N-methylmesoporphyrin IX (NMM) to generate significantly enhanced fluorescence. When DNA MTase is absent or inactive, the DNA probe is stable and cannot release the primers for RCA reaction. In the proposed strategy, the action of DNA MTase on one DNA probe is converted to the multiple amplifications triggered by multiple released primers. The detection limit for Dam MTase is down to 0.0085 U/mL, and the target MTase can be well discriminated from its MTases analogues. The method is utilized in screening of Dam MTase inhibitors and analyzing of spiked Dam MTase in biological samples. The results suggest that the strategy may provide a promising tool for DNA MTase activity detection in biomedical research and cancer theranostics.

A microchip electrophoretic assay for DNA methyltransferase activity based on methylation-sensitive endonuclease Dpnâ ¡.

DNA methylation is a significant epigenetic modification and the methods for the detection of DNA methyltransferase (MTase) activity are important due to aberrant methylation closely related to the occurrence of cancer. In this study, a simple and rapid microchip electrophoresis (ME) coupled with LED-induced fluorescence (LEDIF) method was presented for the detection of Dam MTase activity. This strategy was based on methylation-sensitive endonuclease DpnⅡ which could recognize the same specific site 5'-GATC-3' with Dam MTase in double-stranded DNA (dsDNA). The adenines in the specific site could be methylated by Dam MTase, then the special site could not be digested by DpnⅡ. Both methylated dsDNA and unmethylated dsDNA could be analyzed by ME-LEDIF after stained by SYBR gold. The results showed the fluorescence intensities of methylated dsDNA were directly proportional to Dam MTase activities in the range of 0.5-20 U/mL with a detection limit of 0.12 U/mL. Furthermore, the method could successfully be applied to evaluation experiments of Dam MTase inhibitors. The results confirmed the ME-LEDIF method is a promising approach for inhibitors screening of DNA MTase and development of anticancer drugs.